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Egypt. J. Agric. Res., 96 (1), 2018

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EFFECT OF IRRADIATION AND HEAT TREATMENTS ON ANTIOXIDANT ACTIVITY AND MICROBIAL

LOAD IN CURCUMA (curcuma longa)

GADO BAKR AHMED GADO

Food Tec. Res. Inst., ARC, Giza, Ministry of Agric. Cairo, Egypt.

(Manuscript received 12 September 2017) Abstract

urmeric is the common name used for the dried rhizome of Curcuma longa L. It is a rich source for natural colorants and useful phytochemicals compounds, so that it is used as a

spice and herbal in food processing especially in ready food but it's highly contaminated with pathogenic. Therefore, to find the suitable applied methods for preventing contamination with pathogenic microorganisms without affecting quality. The present work introduced comprehensive studies by using different ordinary treatments as thermal treatments. The investigated treatments included control, thermal treatments at 500C for 15 and 30 min. and the irradiation process doses with 5.0 and 10.0 kGy. The treated samples were evaluated with respect to microbiological and chemical analysis. The obtained results proved that the best ability and suitable method is the gamma irradiation at 10.0 kGy process than other treatments due to its reduce the microbial contamination through activation the antibacterial activity against selected pathogenic microbes such as Staphylococcus aureus, Klebsiella pneumonia, E. coli and Bacillus subtilus. In addition, the same dose was increased the efficiency of antioxidant activity (49%) than other treatments. It was also; found that there was no change in colorant but the fractionation and identification of phenolic and flavonoids compounds by HPLC analysis proved presence of 56 compounds of phenolic and flavonoids which were affected by tested treatments. Irradiation doses were more affecting to generate new major compounds from minor phenols or flavonoids. The advantages of last compounds were increased the antioxidant activity besides increased the antibacterial activity against tested pathogenic microbes. Key words: Turmeric, curcumin, irradiation, antibacterial.

INTRODUCTION

Turmeric rhizome (Curcuma longa L.) is the underground rhizome of Curcuma

long. It is used as a spice, natural colorant and herbal medicine. India is the major

producer and exporter of turmeric. Turmeric rhizomes contained a heavily microbe

load due to being grown under soil surface besides role of environmental factors such

as temperature, humidity, pre/ post-harvesting conditions, and the storage condition

(Araújo and Bauab, 2012). High levels of pathogenic microbes were recorded in food

T



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stuffs mainly as being ‘ready-to-use’. Whereas, no additional further food processing

which can cause out break as recorded before in USA, due to pathogenic bacteria.

Some outbreaks of salmonellosis have been linked to the consumption of foods

seasoned with spices involved turmeric powder. Bacillus cereus may also be present in

spices and herbs, usually at counts below 103 CFU/g but may multiply to high levels

(105 – 106 CFU/g) in food to which it is added; this may be sufficient to cause food

poisoning if the food is inappropriately handled or stored (D’Aoust, 1994).

In addition, the importance of correct food handling practices and usage of

herbs and spices by end users cannot be overemphasized. (Little et al., 2003).

Fumigation with chemicals as ethylene and propylene oxide are being

progressively banned in several countries because of their toxic, mutagenic and

carcinogenic effects (Fowles et al., 2001). While, Lee et al.,( 2005) showed that γ-

irradiation appeared to be the best way for microbial decontamination of spices and

herbs without causing alterations in the condiment quality. The utilization of γ-

irradiation for microbial decontamination of whole spices and spice powders is legally

allowed in more than 50 countries worldwide and it is well established that 40

countries are using this technology commercially (Farag et al., 2013).

The active constituents are the flavonoid curcumin (diferuloylmethane) and

other various volatile oils, including tumerone, atlantone, and zingiberene.

Components of turmeric especially curcumin has been shown to have anti-

inflammatory, antiviral, and anticancer properties (Ji et al.,2012). The literature review

confirmed plethora of information available on safety aspects of curcumin and

essential oil fractions of turmeric (Liju et al., 2012).

While curcuminoids based extracts were well studied for their pharmacological

and safety aspects, polysaccharide extract of turmeric is gaining importance since it

showed to have various pharmacological activities, which include antidiabetic,

antitumour, antidepressant, antioxidant, antimicrobial, antifertility, hepatoprotective,

and immunomodulatory properties (Mohankumar and Farlane,2011). Therefore, the

present work introduced an evaluation on turmeric treated with two methods (thermal

and γ-irradiation) for decontamination, the microbiological, biochemical characteristic

by HPLC profile, antioxidant activity, and coloring ( UV-visible scanning) materials .

MATERIALS AND METHODS

Materials

Turmeric Powder (Curcuma longa L.) rhizomes samples were collected in

sterilized glass bottles from local markets in Cairo, Egypt.Candida albicans (CA),


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Aspergillus niger (AN), as well as four bacteria species, namely, Gram positive

bacteria, Staphylococcus aureus (SA), and Bacillus subtilus (BS), Gram negative

bacteria , Klebsiella pneumoniae (KP), and Escherichia coli (EC) were obtained from

the Egyptian Microbial Culture Collection, (EMCC), Cairo Microbiological Resources

Center (Cairo MIRCEN), Faculty of Agriculture, Ain Shams University, Egypt.

Chloramphenicol,Trimethoprim/ sulphamethnd oxazole, adimethyl sulfoxide (DMSO)

solvent were purchased from Sigma (St. Louis, MO,USA). Sabouraud dextrose agar

and trypticase soya agar medium were purchased from Oxoid Laboratories, UK.

Thermal and Irradiation of samples

The collected turmeric powders (as one replicate=250 g.) were thermally

treated at 500C for 15 and 30 min. Meanwhile, Irradiation treatment were carried out

at doses of 5.0 and 10.0 kGy using a 60Co Russian gamma chamber, (dose rate 1.3

kGy/h), belonging to Cyclotron Project, Nuclear Research Center, Atomic Energy

Authority, Cairo, Egypt.

Microbiological analysis

Total bacterial counts (CFU/g) were determined according to Farag et al.

(2013). Total fungi counts (CFU/g) were conducted according to the method of

Ichinoe Zirnstein and Rehberger. (1983).

Antimicrobial activity of turmeric alcohol extract

In –vitro antimicrobial screening of turmeric alcohol extract was prepared

according to Pundir and Jain (2010). Antimicrobial activity was carried out using the

agar disc diffusion method; culture of strains Candida albicans and Aspergillus niger

were sub-cultured on Sabouraud dextrose agar, as well as four bacteria species, Gram

positive bacteria, namely, Staphylococcus aureus (strains), and Bacillus subtilus, Gram

negative bacteria , Klebsiella pneumoniae, and Escherichia coli were sub-cultured on

Trypticase soya agar medium. Chloramphenicol and Trimethoprim/ sulphamethoxazole

antibacterial agent were used as references to evaluate the potency of the tested

compounds under the same conditions. (Hossain et al., 2012).

Chemical analysis

Total phenol content (TPC) was determined as described by Swain and Hillis

(1959) and later modified by Singleton and Rossi (1965). Antioxidant Activity was

investigated with 2, 2’-Diphenyl-1-picrylhydrazyl method (DPPH) as described by Miller

et al., (2000). Color changes of irradiation or thermal treatments were determined

with spectrophotometric analysis in (1%) Turmeric aqueous solution before and after

treated samples. The mixtures were centrifuged at 10 g for 15 min, and the clear



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solutions were scanned by UV-Visible absorbance measurements at different

wavelength of maximal absorption. A UV/visible spectrophotometer unicam 8625

(Japan) was used. A calibration plot constructed with diluted solutions of tested

sample was employed. The degree of color degradation was calculated as (cb _ ca)x

100/cb, where cb are untreated sample, ca after treated (Farag et al., 2013).

HPLC analysis of phenolic and flavonoid Compounds. The polyphenol components of turmeric were determined by HPLC according

to the method described by Goupy et al., (1999). (1-3 ml) sample after preparation

was collected from the previous step in a vial for injection into HPLC Hewllet packared

(series 1050) equipped with auto-sampling injector, solvent degasser, ultraviolet (UV)

detector was set at 280 nm and quarter HP pump (series 1050). Retention time and

peak area (%) were used to identify and calculate the phenolic compound

concentrations by standard curve of specified concentration. The same instrument

was used, also, to determine the flavonoids compounds of Turmeric. After sample

preparation (1-3 ml) was collected from the previous was step in a vial for injection

into a HPLC Hewllet Packared (series 1050) equipped with auto-sampling injector,

solvent degasser, ultraviolet (UV) detector set at 330 nm and quarter HP pump (series

1050). The column temperature was maintained at 35°C. Retention time and peak

area (%) were used to identify and calculate the flavonoids concentrations by

standard curve of specified concentration.

RESULTS AND DISCUSSION

There is a growing interest in the development and evaluation of natural

antioxidants from medicinal plant materials in the food industry and the field of

preventive health care. Therefore, the hygienic view was applied to find the

appreciate method for decontamination without affecting the quality of spices.

Effects of the thermal and irradiation treatments on microbial load of turmeric

Bacteria of public health importance, such as pathogenic, may be present

and could potentially create a public health risk depending on the end use, such as

addition to ready-to-eat foods that undergo no further processing. Some outbreaks of

Salmonellosis have been linked to the consumption of turmeric powder (Little et al.,

2003). The obtained results in Table (1) proved the presence of bacteria and fungi

was in high levels in control samples. Natural levels of bacteria near 90 X 106 and

8x105 for fungi are logic due to many observations confirmed that the turmeric are

microorganisms highly contaminated herbs. Its bacterial level reaches to 80 to 100

million per gram level (Farkas, 1988). It showed that 96 % of tested turmeric samples


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have aerobic plate count more than 105 /g. This due to its growth under soil surface

besides role of environmental factors such as temperature, humidity, pre-/post-

harvesting conditions and the storage condition (Araújo and Bauab, 2012; Parveen et

al., 2014). Whereas, investigated treatments were reduced these levels mainly γ-

irradiation, i.e., irradiation at low dose (5.0 kGy) was reduced 3 log from 6 log of

bacteria and 2 Log from 5 Log. of fungi at untreated samples. It was found that the

high dose (10.0 kGy) was more effective. Whereas the thermal treatments either at

long or short time at 50 0C was less effective in efficiency than irradiation for

decontamination of turmeric. These results are in parallel with those obtained by

some workers (Zaied et al.,1996; Lee, 2004) who reported that irradiation dose of 3.0

kGy without heat treatment reduced the microbial load in coriander, cumin, turmeric

and chili from 6 log to 3 log and from 5 log to 2 log units depending on the storage

temperature.

Table 1. Effect of thermal and irradiation treatments on microbial load of tested turmeric

Treatments Total bacterial count (CFU/g) Total Fungi count (CFU/g)

Control 610X90 510x8

Thermal process (50⁰C)

-Time 15 min.

-Time 30 min.

410X80

410x 20

410X8.0

410x 6.0

Gamma irradiation

- 5.0kGy.

-10.0kGy

310x30

210x20

310x3.0

4.0x10

Effect of heat and irradiation treatments on antimicrobial activity of

turmeric

In the present work ethyl extract was chosen rather than other solvents due

to its potential as showed by many workers (Pundir and Jain 2010). The obtained

results of antimicrobial activity (AA) of tested turmeric alcohol extracts against yeast,

fungi, and bacterial species are investigated by disc-diffusion assay in Table (2). It is

shows that AA of ethanol extracts of treated turmeric produced inhibition zones

against Staphylococcus aureus, Klebsiella pneumonia, E.coli and Bacillus. Whereas,

no effect was recorded against fungi or yeast from turmeric extract either treated or

not.



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Table 2. Antimicrobial activity of treated turmeric ethyl extracts as inhibition diameter zones size (mm)

Strains

Treatments Control Thermal process

(50⁰C) Gamma

irradiation Positive control

15 min 30 min 5.0 kGy

10.0 kGy

Chloramphenicol T/ S

DMSO

Yeast C. albicans N.R N.R N.R N.R N.R 29 24 N.R

Fungi A. niger N.R N.R N.R N.R N.R 20 18 N.R

Gram(+) Staph. aureus

11 15 17 17 24 25 13 N.R

B. subtilus 9 10 11 15 25 30 10 N.R Gram(-)13

K. pneumonia

9 19 16 12 26 24 23 N.R

E. coli 10 10 11 13 25 24 12 N.R N.R= no response T/ S: Trimethoprim/ sulphamethoxazole DMSO: Dimethyl sulfoxide

Antimicrobial activity of the untreated turmeric extract was observed by

(Niamsa and Sittiwet ,2009). The explanation of antimicrobial activity enhancement

after thermal or irradiation process due to high content of phenolic and flavonoid

which resulted after processing. This phenomenon in food after different process was

observed by workers (Horváthová, 2007; Naguib et al., 2012; Farag et al., 2014).

Effect of thermal and irradiation treatments on total phenols contents and

antioxidant activity of turmeric. Data in Table (3) and Fig.(1) show that, there are slight change in total

phenols contents in all treatments. Meanwhile, the antioxidant activity increased in

both thermal and Gamma irradiation treatments compared to control sample. The

high percent of increase in antioxidant activity was observed after using high dose of

Gamma irradiation (10.0 kGy) was 49 % but less value was resulted in the low dose

of Gamma irradiation (5.0 kGy) was 20 %. A little increase was also, resulted by

thermal treatments. Same results was obtained by (Zaied et al., 1996 ). Table 3. Effect of thermal and irradiation treatments on total phenols contents and

antioxidant activity of turmeric.

*AA -increase (%) of AA=(AA after treatment-AA before/AA before) x100

Treatments Total phenols (mg/g)

Antioxidant activity %

Increasing %*

Control 11.12 29.72

Thermal process(50⁰C ) -Time 15 min. -Time 30 min

10.70 10.65

36.48 39.72

23 34

Gamma irradiation - 5.0 kGy -10.0 kGy

11.05 10.77

35.67 44.19

20 49


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T2 T1 R2 R1 control

05

10152025303540

45

Total phenols (mg/g)

Antioxidant activety (%)

Fig. 1. Effect of thermal and irradiation treatments on total phenol contents and

antioxidant activity of turmeric (T1 and T2 = thermal treatments at 50ºC for 15 and 30 min, respectively. R1 and R2= γ-irradiation doses 5.0 and 10.0 KGy, respectively.

UV-Visible scanning of treated turmeric extract For many centuries, turmeric has been used not only as a spice but also, as a

natural colorant. The bright yellow powder of the ground rhizomes being one of the

earliest known vegetable dyes. This yellow color results from the presence of a

pigment called ‘curcumin’ which may be as high as 5-6% (Akram et al., 2010). No

wonder it is now known as the ‘Yellow Wonder’ The deep orange-yellow powder

known as turmeric is prepared from boiled and dried rhizomes of the plant. Alcohol

extracts scanning using UV-Scanning spectrophotometer, as shown in Fig. (2).

Spectrum of all tested treated extracts. No higher changes were observed in the

spectrum of extract of tested samples, except irradiated samples at its values were

raised may be due to degradation of some compounds (Wang et al,. 2009 ).

Fig. 2. UV-Visible scanning of treated turmeric extract



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HPLC analysis of phenolic and flavonoids compounds of treated turmeric

samples

HPLC analysis of turmeric extract proved that of 56 compounds of phenolic,

flavonoids were prevented and affected by tested treatments as shown in Table

(3).Like same compounds obtained by Shiyou et al., (2011). The tabulated

compounds involved, monoterpenes, sesquiterpenes, fatty acid, steroid besides

different biochemical like volatile oils. The compounds can be classified to majore

(with total area more than 5%) and minor compounds less than 5%. The major as

isochroman (Rt=4.89) and eucalyptol (Rt=8.015), but the minor present the big

percent as shown in Fig. (3). Gamma irradiation doses only were more affecting on

all these compounds, i.e. the major one as isochroman (Rt=4.89 ) in control samples

which occupied (15.54%) increased due to γ-irradiation to 21.85% and 20.74% after

irradiation with 5.0 and 10.0 kGy respectively, as shown in Table (3) and Fig.(3). Also,

eucalyptol (Rt=8.015) with 9.39 % then decreased by all treatments, i.e. 5.93 and

0.59 % for short and long time for thermal treatments. Whereas, irradiation was

more destructive same compound to 0.3% ,0.93 after irradiation with 5.0 and 10.0

kGy, respectively as shown in Table (3) and Fig.(3). Radio-degeneration lead to small

molecular terpens, mono terpens as volatile oils which increased the total area of

these minor compounds. The total of calculated (%) identified phenols and flavonoids

were increased by irradiation to 137 and 131 % due to irradiation with 5.0, 10.0 kGy

respectively as shown in Table (3) and Fig. (3).The decomposition happen by

irradiation or thermal treatments was usually observed by workers due to high energy

which decompose some sensitive terminal groups as–OH then the major compound

can produce series of low carbon number compounds less than 5% as shown in

Fig.(3). The enhancement of phenols and flavonoids by different food processing

especially by irradiation was observed by workers (Horváthová,2007, Naguib et al.,

2012; Farag et al., 2014).


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Table 3. HPLC analysis of treated turmeric. Irradiation treatments

(kGy)

Thermal treatment (time, minute)

Control Compound R t No.

10.0 5.0 30 15 20.74 21.85 16.94 15.54 15.37 Isochroman 4.98 1

1.82 3.62 1.81 1.55 1.58 Coumarin 5.3 2 2.02 5.77 0.4 0.22 0.28 Cumic acid 5.7 3 1.23 1.7 0.25 0.47 0.25 Methyl cumate 6.16 4 0.61 1.33 2.32 1.07 1.37 2,5-Dimethyl-para-anisaldehyde 6.26 5 0.8 0.51 0.3 0.45 0.28 cis-Edulan 6.61 6 1.36 0.95 0.26 0.18 0.19 Dimethoxydurene 6.89 7 0.27 0.29 0.17 0.26 0.21 3,5-Dimethoxycinnamic acid 7.055 8 0.38 0.3 0.67 0.71 1.51 Esculetin 7.2 9 1.14 0.36 0.23 0.19 0.37 Hexestrol 7.42 10 0.37 0.34 0.47 0.26 0.81 β-Myrcene 7.55 11 0.51 0.29 0.18 0.33 0.26 α-Phellandrene 7.7 12 0.3 0.45 0.29 0.17 0.16 p-Cimene 7.9 13 0.93 0.29 0.59 5.93 9.39 Eucalyptol 8.015 14 0.35 0.37 0.2 0.17 0.25 Daidzein 8.39 15 0.32 0.75 0.23 0.16 0.61 Terpinolene 8.65 16 0.54 0.32 0.84 0.6 0.61 Sinapic acid 9.12 17 0.45 4.19 1.14 0.45 0.32 Nabilone 9.46 18 5.46 2.36 0.24 0.21 0.21 Fisetin 9.7 19

3.27 0.51 0.49 0.18 0.41 Chromone, 5-hydroxy-6,7,8-trimethoxy-2,3-dimethyl- 10.7 20

0.81 1.35 0.41 0.34 0.68 β Ionol 11.38 21 2.21 0.29 0.22 0.56 0.22 geranyl-α-terpinene 11.78 22 0.24 0.79 0.47 0.49 0.42 α-Curcumene 12.11 23 0.26 0.57 3.49 2.83 2.97 trans-Nuciferol 12.3 24 1.04 1.32 2.28 2.06 1.95 α-Zingiberene 12.4 25 2.17 0.8 0.24 0.35 0.66 α-Ylangene 12.45 26 0.42 0.33 1.66 1.33 2 cis-Farnesol 12.566 27 23.08 28.01 0.29 0.42 0.28 cis-Thujopsene 12.685 28 0.81 0.6 0.37 0.75 0.39 (-)-Spathulenol 12.889 29 5.56 4.33 0.19 0.3 0.21 9-Methoxycalamenene 12.97 30 0.45 0.35 1.83 1.37 1.67 Germacrene 13.18 31 1.34 1.25 0.25 0.46 0.2 β-Himachalene 13.28 32 1.07 0.51 0.73 0.39 0.7 δ-Neoclovene 13.47 33 2.42 1.31 1.73 1.43 1.41 Caryophylene oxide 13.55 34 1.06 0.59 7.42 6.79 6.6 β-Gurjunene 13.62 35 0.81 0.96 5.51 3.63 3.05 Palustrol 13.69 36 0.77 0.67 2 1.56 1.48 β-Santalol 13.85 37 0.42 0.42 4.66 3.34 3.14 γ-Elemene 13.93 38 2.55 2.14 2.2 0.68 1.9 δ-Tocopherol 14.17 39 0.43 0.28 1.48 0.67 1.39 (S)-(-)-Citronellic acid 14.24 40 1.68 1.13 3.4 2.05 1.98 Z)-trans- α-Bergamotol 14.38 41 5.89 3.24 0.85 0.71 1.03 Squalene 14.44 42 1.2 1.53 2.16 1.79 2.37 Sclareol 14.56 43 0.43 0.66 1.34 0.21 0.48 p-Menth-1-en-3-one 15.27 44 20.74 21.85 1.57 0.28 1.24 Camphoric acid 15.42 45 1.82 3.62 1 1.38 1.08 Digitoxin 15.6 46 2.02 5.77 4.92 4.46 5.26 Octadecanoic acid 15.7 47 1.23 1.7 1.01 0.72 1.51 Zearalenone 15.8 48

0.61 1.33 4.26 4.46 4.18 Linolenic acid 16.86 49 0.8 0.51 8.47 7.08 3.87 Lupulon 16.96 50 1.36 0.95 1.68 2.2 1.65 cis-Vaccenic acid 17.1 51 0.27 0.29 0.95 1.51 2.01 D-Glucuronic acid 18.9 52 0.38 0.3 1.19 0.54 1.91 7,8-Dihydro-α-ionone 20.28 53 1.14 0.36 0.48 0.36 0.87 methyl α-curacinoside 22.298 54 0.37 0.34 0.42 0.92 1.92 Lutein 23.11 55 0.51 0.29 0.84 12.47 2.89 Rhodovibrin 23.6 56



144

0102030405060708090

Control

Thermal-

15

Therm

al-30

Irrad

.5kGy

Irrad

.10kG

y

Treartments

Con

cent

ratio

n (%

) More than 5% Less than 5%

Fig. 3. Effect of treatments on phenols and flavonoids compounds (%).

CONCLUSION

Finally, it could be clearly concluded through this study, this work that has

technological and applicable values, as it proved the significance of turmeric as a

natural colorant and also as a spice preferred by the majority of consumers. Besides,

irritation was an effective procedure to increase the antioxidant activity substances of

turmeric as well as inhibiting the activity of pathogenic bacteria. It was also proved

that irradiation had no harmful effect on coloring capacity of turmeric.

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Nutrition & Food Research, 40(1):32-36.


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مضاداتعلى والمعامالت الحرارية التشعيعتأثير الحمل الميكروبى للكركمواالكسدة

جادو بكر أحمد جادو

مصر –وزارة الزراعة –مركز البحوث الزراعية بالجيزة –معهد بحوث تكنولوجيا االغذية

عديد من االستخدامات فى .Curcuma longa L هو االسم الشائع للريزوم لنبات والكركم يستخدم

يستخدم على فهو لذا، وملونات طبيعيةهامة كيماوية مركبات حتوى علىه يالتكنولوجيه، حيث ان اال أنهاالغذية الجاهزة، خصوصاابل وملون طبيعى فى االغذية ونطاق واسع فى التصنيع الغذائى كت

. ولهذا السبب فانه من الضرورى ايجاد طريقة الممرضهعالى التلوث الميكروبى خاصة الميكروبات تاثير على جودته وقد تم تنفيذ العمل الحالى لدراسة مقارنة باستخدام طرق من غير لتعقيم الكركم

باستخدام اشعة جامادقيقة) والتشعيع ٣٠، ١٥م لمدة °٥٠طبيعية مثل الحرارة المنخفضة (التغيرات الميكروبيولوجية على كيلو جراى) بجانب الكنترول (غير المعامل) ١٠، ٥بجرعات (كيلو جراى أعطى أفضل النتائج فى ١٠االشعاع على الجرعه أثبتت النتائج ما يلى: وقدوالكيماوية.

إزالة البكتريا وتقليل الفطريات وكذلك فى تشجيع النشاط المضاد للميكروبات ضد انواع من البكتريا %٤٩ت زيادة محتوى المواد المضادة لالكسدة بنسبة بلغ كذلك مقارنة بالمعاملة الحرارية والكنترولعدم حدوث أى أضرار فى المواد الملونه عند التشعيع بجرعة و من المحتوى الكلى للفينوالت الكلية

مركب من ٥٦ماتوجرافى وجود والكنترول. وقد ثبت بالتحليل الكربمثل مقارنةي كيلو جرا ١٠أكثر فاعلية الفينوالت والفالفونيدات حيث تكسرت المركبات الكبيرة واعطت مركبات فينولية عديدة

كيلو جراى للحفاظ على جودة الكركم مع تقليل الحمل ١٠وهو ما يعطى التوصية باستخدام الجرعة الميكروبى.

EFFECT OF IRRADIATION AND HEAT TREATMENTS ON …§لبحث الرابع... · essential oil...

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